Cysteine-674 oxidation and degradation of sarcoplasmic reticulum Ca2+ ATPase in diabetic pig aorta

Abstract: The sarcoplasmic reticulum Ca2+ ATPase (SERCA) is redox-regulated by posttranslational thiol modifications of cysteine-674 to regulate smooth muscle relaxation and migration. To detect oxidation of cysteine-674 that irreversibly prevents redox regulation, a polyclonal, sequence-specific antibody was developed toward a peptide containing cysteine-674 sulfonic acid. The antibody stained intact 110-kDa SERCA in pig cardiac SR that was oxidized in vitro by peroxynitrite in a sequence-specific manner, and histochemically stained atherosclerotic pig and rabbit aorta. Surprisingly, immunoblots of the pig aorta failed to stain intact 110-kDa SERCA protein, but rather, higher molecular mass aggregates and lower molecular mass bands. Of the latter bands at 70 and 60 kDa, the largest were observed in diabetic, hyperlipidemic pigs, and coincided with the most positive histochemical staining. The 70- and 60-kDa molecular mass bands also coincided with the majority of the protein detected by a monoclonal total anti-SERCA antibody, which detected the intact 110-kDa protein in normal pigs. Mass spectrometry identified SERCA in all the major bands detected by the sulfonic acid antibody as well as the oxidation of cysteine-674 in the 70-kDa band. These studies demonstrate a sequence-specific antibody that detects partial degradation products of SERCA, which represent the majority of the protein in some diabetic hypercholesterolemic pig aortae. In addition, the results suggest an association between irreversible oxidation of SERCA and its degradation, and that an important portion of the oxidized protein in tissue samples may be partially degraded. [Copyright &y& Elsevier]